Organophosphorous Compounds for the Activation of Gamma/Delta T Cells

ABSTRACT

The present invention describes organophosphorus compounds of general formula (I) 
     
       
         
         
             
             
         
       
     
     their preparation and their uses in the activation of gamma/delta T-cells, in the screening of GcpE and LytB enzyme inhibitors and in the prophylaxis and treatment of diseases in humans and animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/137,241, filed on Jun. 11, 2008, now U.S. Pat. No. 7,871,992 which isa continuation of U.S. application Ser. No. 10/484,143, filed on Oct.22, 2004, now U.S. Pat. No. 7,399,756, which is the national stage ofinternational application No. PCT/EP02/07986, filed Jul. 18, 2002, whichare incorporated herein by reference in their entireties, including allreferences, Figures, Tables and nucleic acid and polynucleotidesequences.

Numerous diseases in humans and animals are caused by the abnormalfunctioning of the immune system. Consequently, there is a high demandfor substances that are able to regulate the immune system.

It is known how to employ the classical acetate/mevalonate pathway inthe biosynthesis of isoprenoids (Beycia E D, Porter J W, Annu RevBiochem. 1976; 45:113-42), and an alternative method of biosynthesis isknown that is independent of mevalonate, namely the2-methyl-D-erythritol pathway (MEP, synonymous with DOXP) (Rohmer M. NatProd Rep. 1999 October; 16(5):565-74). Both pathways lead to isopentenylpyrophosphate (IPP), the common precursor of all higher isoprenoids.While the acetate/mevalonate pathway has been known for a long time andhas been thoroughly explained, not all biosynthetic reaction steps thatoccur along the MEP are as yet known.

It is known that human gamma/delta T-cells are activated by one orseveral intermediates along the MEP. This means that a selectiveproliferation and cytokine secretion of the gamma/delta T-cellpopulation is brought about during the incubation of peripheral bloodlymphocytes with extracts from organisms that possess the MEP (Jomaa H,Feurle J, Luhs K, Kunzmann V, Tony H P, Herderich M, Wilhelm M, FEMSImmunol Med Microbiol, 1999 September; 25(4):371-8). The exact chemicalcomposition of this/these activating substance(s) is still unknown. Thepublished data indicate that 3-formyl-1-butyl pyrophosphate is formed asa hypothetical intermediate of the MEP and that it plays a role in theactivation of gamma/delta T-cells (Belmant C, Espinosa E, Poupot R,Peyrat M A, Guiraud M, Poquet Y, Bonneville M, Fournie J J, J Biol.,Chem, 1999 Nov. 5; 274(45):32079-84).

The object of this invention is to provide substances which are able tostimulate gamma/delta T-cells and thereby have a regulatory effect onthe immune system.

This object is achieved by medicines which contain one or more of thesubstances defined in Claim 1 as well as in the subordinate claims.

Surprisingly, it has been found that compounds of formula (I) areeminently suitable for the activation of gamma/delta T-cells.

wherein R₁ is selected from the group comprising a methyl residue, aformyl residue, substituted and unsubstituted hydroxymethyl residues andC₀H₂R₃₁, wherein R₃₁ is selected from the group comprising OH,substituted and unsubstituted phosphate and substituted andunsubstituted pyrophosphate and R₃₁ and R₂ cannot be present in themolecule at the same time,

R₃₃ is selected from the group comprising hydrogen, OH, substituted andunsubstituted phosphate and substituted and unsubstituted pyrophosphate,

R₃ is selected from the group comprising hydrogen, substituted andunsubstituted alkyl with 1 to 26 carbon atoms, substituted andunsubstituted hydroxyalkyl with 1 to 26 carbon atoms, substituted andunsubstituted aryl, substituted and unsubstituted aralkyl. substitutedand unsubstituted alkenyl with 1 to 26 carbon atoms, substituted andunsubstituted alkinyl with 1 to 26 carbon atoms, substituted andunsubstituted cycloalkyl, substituted and unsubstituted heterocyclicresidues, substituted and unsubstituted phosphate, a silyl, anucleoside, a nucleoside mono-, di- or triphosphate, a deoxynucleoside,a cation of an organic or inorganic base, particularly a metal of thefirst, second or third main group of the Periodic Table, ammonium,substituted ammonium and ammonium compounds derived from ethylenediamineor amino acids, and OR₃₄, wherein R₃₄ is defined like R₃,

X₂, inasmuch as a ring is formed between X₂ and C₁, is defined like X₁,and otherwise X₂ is selected from the group comprising —OR₆,

wherein R₇ and R₈ are defined like R₃₄,

wherein R₄ is defined like R₃, and Z₁ is defined like X₁, and X₃, if itforms a ring with C₁, is defined like X₁ and, if does not form a ringwith C₁, corresponds to a group

wherein R₅ is defined like R₃, and Z₂ and X₄, which forms a ring withC₁, are defined like X₁,

R₂ is selected from the group comprising hydrogen, OH, alkoxy, phenoxy,benzylo substituted and unsubstituted phosphate and substituted andunsubstituted pyrophosphate,

X₁ can be oxygen or

wherein Y₁ and Y₂ can be the same or can be different and are selectedfrom the group comprising H, OH, halogen, an amino residue, aC₁₋₉-alkoxy residue and a C₁₋₉-alkylthio residue, or together form anoxo group,

and a double bond can be present between C₀ and C₁, or between C₁ andC₂, or between C₂ and C₃.

Preference is given to compounds having the formula:

wherein a single or double bond is present between C₂ and C₃, R₁ isselected from the group comprising a methyl residue, a formyl residueand substituted and unsubstituted hydroxymethyl residues,

R₂ is selected from the group comprising hydrogen, hydroxyl, alkoxy,phenoxy and benzyloxy residues, substituted and unsubstituted phosphateand substituted and unsubstituted pyrophosphate,

X₁ is oxygen or corresponds to a group

wherein Y₁ and Y₂ can be the same or can be different and are selectedfrom the group comprising H, OH, halogen, amino and C₁₋₉-alkoxy andC₁₋₉-alkylthio residues, or together form an oxo group,

R₃ is selected from the group comprising hydrogen, substituted andunsubstituted alkyl with 1 to 26 carbon atoms, substituted andunsubstituted hydroxyalkyl with 1 to 26 carbon atoms, substituted andunsubstituted aryl, substituted and unsubstituted aralkyl, substitutedand unsubstituted alkenyl with 1 to 26 carbon atoms, substituted andunsubstituted alkinyl with 1 to 26 carbon atoms, substituted andunsubstituted cycloalkyl, substituted and unsubstituted heterocyclicresidues, substituted and unsubstituted phosphate, a silyl, anucleoside, a nucleoside mono-, di- or triphosphate, a deoxynucleoside,a cation of an organic or inorganic base, particularly a metal of thefirst, second or third main group of the Periodic Table, ammonium,substituted ammonium and ammonium compounds derived from ethylenediamineor amino acids,

X₂, inasmuch as a ring is formed between X₂ and C₁, is defined like X₁,and otherwise X₂ corresponds to

wherein R₄ is defined like R₃, and Z₁ is defined like X₁, and X₃, if itforms a ring with C₁, is defined like X₁ and, if it does not form a ringwith C₁, corresponds to a group

wherein R₅ is defined like R₃, and Z₂ and X₄, which forms a ring withC₁, are defined like X₁.

Particular preference is given to compounds having formula (IIA)

wherein C₂ and C₃ are linked together by either a single or a doublebond, R₁ is a methyl group or a substituted or unsubstitutedhydroxymethyl group, R₂ is hydrogen, OH, a substituted or unsubstitutedphosphate or a substituted or unsubstituted pyrophosphate, X₁ and X₂ areselected from the group comprising O, CHF, CHCl, CFCl, CH₂, CF₂ or CCl₂,and R₃ is selected from the group comprising hydrogen, substituted andunsubstituted phosphate, a nucleoside, a nucleoside mono-, di- ortriphosphate, a deoxynucleoside, a cation of an organic or inorganicbase, particularly a metal of the first, second or third main group ofthe Periodic Table, ammonium, substituted ammonium and ammoniumcompounds derived from ethylenediamine or amino acids.

Preference is also given to compounds having formula (IIB)

wherein C₂ and C₃ are linked together by either a single bond or adouble bond, R₁ is a methyl group or a substituted or unsubstitutedhydroxymethyl group, R₂ is H if R₁ is a substituted or unsubstitutedhydroxymethyl and is OH, a substituted or unsubstituted phosphate or asubstituted or unsubstituted pyrophosphate if R₁ is a methyl residue,X₁, X₂ and X₃ are selected from the group comprising O, CHF, CHCl, CFCl,CHl₂, CF₂ or CCl₂, and R₃ and R₄ are selected from the group comprisinghydrogen, substituted and unsubstituted phosphate, a nucleoside, anucleoside mono-, di- or triphosphate, a deoxynucleoside, a cation of anorganic or inorganic base, particularly a metal of the first, second orthird main group of the Periodic Table, ammonium, substituted ammoniumand ammonium compounds derived from ethylenediamine or amino acids.

Preference is likewise given to compounds having formula (IIC)

wherein a single or double bond can be present between C₂ and C₃, R₁ isa methyl or a substituted or unsubstituted hydroxymethyl group, R₂ is H,OH, a substituted or unsubstituted phosphate or a substituted orunsubstituted pyrophosphate, X₁, X₂, X₃ and X₄ are selected from thegroup comprising O, CHF, CHCl, CFCl, CH₂, CF₂ or CCl₂, and R₃, R₄ and R₅are selected from the group comprising hydrogen, substituted orunsubstituted phosphate, a nucleoside, a nucleoside mono-, di- ortriphosphate, a deoxynucleoside, a cation of an organic or inorganicbase, particularly a metal of the first, second or third main group ofthe Periodic Table, ammonium, substituted ammonium and ammoniumcompounds derived from ethylenediamine or amino acids.

Moreover, preferred compounds having formulae (II) and (IIA) to (IIC)are those wherein R₁ is a substituted or unsubstituted hydroxymethylresidue, particularly hydroxymethyl itself or a hydroxymethyl residuesubstituted by phosphate, diphosphate or nucleoside diphosphate, forexample a hydroxymethyl residue substituted by uridine diphosphate, andR₂=H.

Compounds which are likewise preferred are those having formulae (II)and (IIA) to (IIC) in which R₁ is a methyl residue and R₂ is a hydroxylresidue, a substituted or unsubstituted phosphate residue or asubstituted or unsubstituted diphosphate residue, particularly anucleoside diphosphate residue, e.g. a uridine diphosphate residue.

The following compounds are particularly preferred:

wherein the residues R₃, R₄ and R₅ are selected from the groupcomprising hydrogen, ammonium, sodium or potassium.

Furthermore, preferred compounds also include those having the followingformula:

wherein R₃₁ and R₂, which cannot both be present in the molecule at thesame time, are selected from the group comprising OH, substituted andunsubstituted phosphate and substituted and unsubstituted pyrophosphate;if R₃₁ is present in the molecule, a double bond is formed between C₁and C₂, and a double bond is analogously formed between C₀ and C₁ if R₂is present in the molecule; R₃₃ is selected from the group comprisinghydrogen, OH, substituted and unsubstituted phosphate and substitutedand unsubstituted pyrophosphate; R₃₄ is selected from the groupcomprising hydrogen, substituted or unsubstituted alkyl with 1 to 26carbon atoms, substituted or unsubstituted hydroxyalkyl with 1 to 26carbon atoms, substituted or unsubstituted aryl, substituted orunsubstituted aralkyl, substituted or unsubstituted alkenyl with 1 to 26carbon atoms, substituted or unsubstituted alkinyl with 1 to 26 carbonatoms, substituted or unsubstituted cycloalkyl, a substituted orunsubstituted heterocyclic residue, substituted or unsubstitutedphosphate, a silyl, a nucleoside, a deoxynucleoside, a nucleoside mono-,di- or triphosphate, a cation of an organic or inorganic base,particularly a metal of the first, second or third main group of thePeriodic Table, ammonium, substituted ammonium or ammonium compoundsderived from ethylenediamine or amino acids; X₂ is either —OR₆, whereinR₆ is defined analogously to R₃₄, or may be

wherein R₇ and R₈ are defined like R₃₄; and X₁, X₃₂ and X₃₃ can be thesame or can be different and can be oxygen or a group

wherein Y₁ and Y₂ can be the same or can be different and are selectedfrom the group comprising H, OH, halogen, an amino residue, aC₁₋₉-alkoxy residue and a C₁₋₉-alkylthio residue, or together form anoxo group,

Particularly preferred compounds are those having formula (IIIA)

wherein R₃₁ and R₂, which cannot be present in the molecule at the sametime, are selected from the group comprising OH, substituted andunsubstituted phosphate and substituted and unsubstituted pyrophosphate;if R₃₁ is present in the molecule, a double bond is formed between C₁and C₃, and a double bond is formed analogously between C₀ and C₁ if R₂is present in the molecule; R₃₄, R₇ and R₈ can be the same or can bedifferent and are defined as above; and X₁, X₃₂ and X₃₃ can be the sameor can be different and are defined as for compound (III).

Hydrogen substituents at C₁, C₂ and C₃ are not explicitly indicated informulae (I) to (IIIA) for reasons of clarity. However, carbon atoms areunderstood as being tetravalent. The missing substituents are thereforehydrogen radicals.

Furthermore, preference is given to compounds having formula (IIIA) inwhich R₃₁ and R₂ are either OH or substituted or unsubstitutedphosphate, R₃₄, R₇ and R₈ are selected from the group comprisingsubstituted and unsubstituted phosphate, a nucleoside, adeoxynucleoside, a nucleoside mono-, di- or triphosphate, a cation of anorganic or inorganic base, particularly a metal of the first, second orthird main group of the Periodic Table, ammonium, substituted ammoniumand ammonium compounds derived from ethylenediamine or amino acids, andX₁, X₃₂ and X₃₃ can be the same or can be different and are O, CHF,CHCl, CFCl, CH₂, CF₂ or CCl₂.

Other preferred compounds having formula (IIIA) are those in which thephosphate groups are present as sodium, potassium or substituted orunsubstituted ammonium salts.

The following compounds are most suitable:

Other embodiments of the invention are defined by the subordinateclaims.

Peculiarities of the abovementioned definitions and suitable Examples ofthese will be given below:

“Alkyl” is a straight-chain or branched-chain alkyl residue with up to26 carbon atoms, unless otherwise stated, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the like.

Unless otherwise stated, “alkenyl” includes straight-chain orbranched-chain alkenyl groups with up to 26 carbon atoms, e.g. vinyl,propenyl (e.g. 1-propenyl, 2-propenyl), 1-methylpropenyl,2-methylpropenyl, butenyl, 2-ethylpropenyl, pentenyl and hexenyl.

Unless otherwise stated, “alkinyl” includes straight-chain orbranched-chain alkinyl groups with up to 26 carbon atoms.

Cycloalkyl preferably refers to a C₃-C₇-cycloalkyl that may besubstituted; alkyl, alkoxy (e.g. methoxy, ethoxy, etc.), halogen (e.g.fluorine, chlorine, bromine, etc.), nitro and the like are suitable aspossible substituents.

Aryl is an aromatic hydrocarbon residue, such as phenyl, naphthyl, etc.,which may have one or more suitable substituents such as alkoxy (e.g.methoxy, ethoxy, etc.), halogen (e.g. fluorine, chlorine, bromine,etc.), nitro and the like.

“Aralkyl” includes mono-, di- and triphenylalkyls such as benzyl,phenethyl, benzhydryl, trityl and the like, where the aromatic part mayhave one or more suitable substituents such as alkoxy (e.g. methoxy,ethoxy, etc.), halogen (e.g. fluorine, chlorine, bromine, etc.), nitroand the like.

“Alkoxy residue” relates to a straight-chain or branched-chain alkoxyresidue with up to 26 carbon atoms, such as methoxy or ethoxy residues,etc., unless otherwise stated. It can be substituted for example byhydroxyl, amino, halogen and oxo groups and alkoxy residues such asmethoxy or ethoxy residues.

Unless otherwise stated, “hydroxymethyl residue” relates to a residuethat has a substituted or unsubstituted C₁-C₉-alkyl, aryl or aralkylresidue, e.g. methoxymethyl, ethoxymethyl, phenoxymethyl orbenzoxymethyl, etc., attached to the oxygen, or has a substituted orunsubstituted phosphate or pyrophosphate residue, such as adenosinediphosphate, uridine diphosphate, etc., attached to the oxygen.

Unless otherwise stated, “alkylthio residue” relates to a straight-chainor branched-chain alkylthio residue with up to 9 carbon atoms, such asthiomethyl or thioethyl residues, etc. It can be substituted e.g. byhydroxyl, amino, halogen and oxo groups and alkoxy residues such asmethoxy or ethoxy residues.

“Silyl residues” can, for example, be substituted by the above-definedalkyl residues or cycloalkyl-(C₀₋₂₆)-alkyl residues.

“Silyl-(C₀₋₂₆)-alkyl groups” are silyl residues that can also be bondedto the framework by means of an alkyl residue. The alkyl and silylgroups are defined as above.

The alkane and/or arene parts may, if desired, have at least onesuitable substituent such as a halogen, alkoxy, hydroxyl, nitro or thelike, in the case of the aforementioned esters.

Substituted and unsubstituted phosphate residues or substituted andunsubstituted pyrophosphate residues include salt compounds of thecorresponding phosphoric acid derivatives with organic or inorganicbases (e.g. sodium salt, potassium salt, calcium salt, aluminium salt,ammonium salt, magnesium salt, triethylamine salt, ethanolamine salt,dicyclohexylamine salt, ethylenediamine salt,N,N′-dibenzylethylenediamine salt, etc.), as well as amino acid salts(e.g. arginine salt, lysine salt, glycine salt, alanine salt, ornithinesalt, etc.), and also residues in which the phosphate group forms esterswith substituted or unsubstituted C₁-C₂₆-alkyl, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, substituted orunsubstituted cycloalkyl or a substituted or unsubstituted heterocyclicresidue, or with a nucleoside or a deoxynucleoside. “Nucleoside” isunderstood as meaning adenosine, guanosine, uridine, thymidine andcytidine, while “deoxynucleoside” is understood as meaningdeoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine anddeoxyuridine.

The invention also relates to the pharmaceutical salts and esters of thesalts. Moreover, it includes all spatial isomers of the compounds, bothas pure substances and as mixtures thereof.

The substances according to the invention can be obtained from bacteria,algae, plants and protozoa, including those in which the lytB gene hasbeen deleted, and purified (Example 1). Purification can be effected bymeans of HPLC or by other methods known per se, such as electrophoresis,precipitation (e.g. as a barium salt) or other chromatographictechniques.

Various applications of the compounds are possible. Accordingly, it hasbeen shown, for example, that the substances can be employed in theactivity testing of the enzymes GcpE and LytB, as well as in testsystems for the measurement of gamma/delta T-cell activation (seeExamples 2, 5).

The substances according to the invention can either be chemicallysynthesized (Example 3) or be obtained from bacteria, algae, plants andprotozoa and purified (Example 4). Purification can be effected by meansof HPLC or by other methods known per se, such as electrophoresis,precipitation (e.g. as a barium salt) or other chromatographictechniques.

Furthermore, the substances according to the invention can be used in ascreening procedure for the identification of GcpE and LytB enzymeinhibitors, as they are intermediates of the MEP. This method ofdetermining the activity of the enzymes is based on the measurement ofdifferences in the concentration of the enzyme substrates and productsunder suitable reaction conditions. By bringing suitable test substancesinto contact with the enzymes during activity determination, inhibitorscan be identified by the reduction in the observed enzyme activity. Theinhibitors are suitable as herbicides and as active ingredients withantibacterial, antiparasitic and antiviral activity in humans andanimals.

The compounds according to the invention can also be used in theproduction of medicines. The efficacy of the compounds is based on theactivation of gamma/delta T-cells. Depending on the field ofapplication, the immunological defence mechanism can thereby bestrengthened or an immunological tolerance can be induced towardsautoantigens and allergens.

The fields of application are the treatment of immune and autoimmunediseases and allergies in humans and animals. Examples of these are:allergies, multiple sclerosis, rheumatoid arthritis, Hashimoto'sthyroiditis, myasthenia gravis, lupus erythematosus, diabetes mellitus,primary biliary cirrhosis, active chronic hepatitis,adrenalitis/Addison's disease, polymyositis, dermatomyositis, autoimmunehaemolytic anaemia, myocardial inflammation and inflammation of theheart membrane, scleroderma, uveitis (phacouveitis, sympatheticophthalmia), pemphigus vulgaris, pemphigoid, pernicious anaemia,autoimmune atrophic gastritis, inflammatory diseases of the intestine,such as Crohn's disease and ulcerative colitis, and inflammatorydiseases of the lung, such as asthmatic and bronchitic ailments.

The preferred fields of application are Crohn's disease, ulcerativecolitis, multiple sclerosis, asthma, chronic bronchitis and allergies.

Furthermore, it has been shown that the substances according to theinvention can be successfully employed in the treatment of diseaseswhich are caused by viruses, bacteria and parasites.

In particular, the substances defined in claim 1 and the subordinateclaims are suitable for the prevention and treatment of tumours that arecaused by microorganisms. Bacteria, such as

Helicobacter pylori (e.g. tumours of the gastrointestinal tract), andpapilloma viruses (e.g. tumours of the female genitalia), belong to thisgroup of microorganisms.

The compounds defined in the claims are particularly suitable for theprophylaxis and treatment of one of the aforesaid diseases as well ashepatitis C virus infections and benign and malignant tumours,particularly those caused by papilloma viruses, and for helicobactereradication therapy in cases of ulceration of the gastrointestinaltract.

For medicinal purposes, pharmaceutical preparations can be used on theirown or in combination with other medicines and can contain either theisolated substances according to the invention or living or deadorganisms containing the substances. They are preferably used incombination with substances that are recognized by the immune system asbeing foreign antigens or autoantigens.

Examples of these are myelin basic protein (MBP) and other extracts ofthe tissue of the nervous system, type I, II or III collagen,thyroglobulin, acetylcholine receptor protein, DNA, islet cell extracts,human insulin, liver extracts, hepatocellular extracts, adrenocorticalextracts, skin extracts, heart extracts, muscle extracts, skin cellextracts, haemopoietic line cell extracts, eye lens proteins,S-antigens, S-antigen mixtures, stomach cell extracts, parietal cellextracts, intrinsic factor and intestinal extracts.

Preferred forms of administration are oral, inhalational, intravenous,parenteral. intracisternal, intravaginal, intraperitoneal, local(powder, ointment, drops) and rectal administration, as well asapplication to the skin or mucous membranes.

The invention includes the administration of an inhalant containing atleast one of the substances defined in claim 1 for the treatment ofhuman diseases, particularly allergies and diseases of the respiratorytract such as asthma and chronic bronchitis.

Suitable pharmaceutical compositions are moreover: tablets, retardtablets, dragees, capsules, premixes, pills, pellets, boli, aerosols,granules, suppositories, solutions, concentrates, suspensions andemulsions, pastes, ointments, gels, creams, lotions, powders, infusionsand sprays. The pharmaceutical formulations may correspond to a fractionor a multiple of a single dose. Dosage units can be 1, 2, 3 or 4 times asingle dose, for example, or may contain ½, ⅓ or ¼ of a single dose. Asingle dose preferably contains the quantity of active ingredient whichis used for one administration and which usually corresponds to a whole,a half, a third or a quarter of the daily dosage.

Tablets, dragees, capsules, pills and granules may contain the activeingredients in addition to the usual excipients such as (a) fillers anddiluents, e.g. starches, lactose, cane sugar, glucose, mannitol andsilicic acid, (b) binders, e.g. carboxymethyl cellulose, alginates,gelatine and polyvinylpyrrolidone, (c) humectants, e.g. glycerine, (d)disintegrating agents, e.g. agar-agar, calcium carbonate and sodiumcarbonate, (e) solution retarders, e.g. paraffin, and (f) absorptionaccelerators, e.g. quaternary ammonium compounds, (g) wetting agents,e.g. cetyl alcohol and glycerol monostearate, (h) adsorbents, e.g.kaolin and bentonite, and (i) lubricants, e.g. talcum, calcium andmagnesium stearate and solid polyethylene glycols, or mixtures of thesubstances listed under (a) to (i). Moreover, the compounds according tothe invention can also be incorporated into other carrier materials suchas plastics (plastic chains for local therapy), collagen or bone cement.

The tablets, dragees, capsules, pills and granules may be provided withthe usual coatings and envelopes optionally containing opaquing agents,and can be prepared in such a way that the active ingredients arereleased, optionally with a delay, only in the intestinal tract or,preferably, in a particular part of the intestinal tract, it beingpossible to use e.g. polymer substances and waxes as embeddingcompounds.

The active ingredients can also be in microencapsulated form, optionallywith one or more of the aforesaid excipients.

In addition to the active ingredients, suppositories may contain theusual water-soluble or water-insoluble excipients, e.g. polyethyleneglycols, fats, e.g. cacao fat, and higher esters (e.g. a C₁₄ alcoholwith a C₁₆ fatty acid), or mixtures of these substances.

In addition to the active ingredient(s), ointments, pastes, creams andgels can contain the usual excipients, e.g. animal and vegetable fats,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acids, talcum andzinc oxide, or mixtures of these substances.

In addition to the active ingredient(s), powders and sprays may containthe usual excipients, e.g. lactose, talcum, silicic acid, aluminiumhydroxide, calcium silicate and polyamide powder, or mixtures of thesesubstances. Additionally, sprays may also contain the usual propellant,e.g. chlorofluorocarbons.

In addition to the active ingredients, solutions and emulsions maycontain the usual excipients such as solvents, solubilizers andemulsifying agents, e.g. water, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils, particularlycottonseed oil, peanut oil, maize oil, olive oil, castor oil and sesameoil, glycerine, glycerol formal, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitol, or mixtures ofthese substances.

There are very marked differences in the amounts of the individualderivatives that are necessary in order to achieve the desired effect.Generally speaking, both in human as well as in veterinary medicine, ithas proved to be advantageous if the active ingredient(s) of formula (I)are administered in total amounts of approximately 0.01 to about 2000 μgevery 24 hours, if necessary in the form of several single doses, inorder to achieve the desired results. A single dose preferably containsthe active ingredient(s) in amounts of approximately 0.01 to about 2000μg. However, it may be necessary to deviate from the abovementioneddosages, depending on the type and body weight of the person to betreated, the nature and severity of the disease, the type of preparationand the administration of the medicine, as well as the period orinterval over which the preparation is administered.

Consequently, it may be sufficient in a number of cases to manage withless than the abovementioned quantity of active ingredient, while inother cases the aforementioned quantity of active ingredient will haveto be exceeded. The optimum dosage required and the type ofadministration of the active ingredients can be determined by thoseskilled in the art on the basis of their specialist knowledge.

In the treatment of animals, the compounds to be used according to thisinvention can be given in the usual concentrations and preparationstogether with the food or food preparations or with the drinking water.

EXAMPLE 1 Purification of Gamma/Delta T-Cell Activating Compounds

Various gamma/delta T-cell activating compounds were isolated fromCoryne-bacterium ammoniagenes. 28 kg of the cell mass were digested witha Dynax mill in 50 mM ammonium formate buffer (pH 8.0). Afterpreabsorption on a hydrophobic polystyrene matrix, the digested materialwas loaded onto an anion exchanger and eluted with a stepped gradient(100, 300, 500 mM ammonium formate, pH 8.0). The 300 mM eluate waspassed through a C-18 matrix and then through a 3 kDa hollow fibrefilter for ultrafiltration. The filtrate was diluted with water to 30 mMammonium formate and loaded once more onto an anion exchanger. Elutionthen took place with a linear gradient of 30 to 500 mM ammonium formate.Individual fractions were tested for their ability to activategamma/delta T-cells. Then some of the active compounds were precipitatedas barium salts by the admixture of 100 mM BaCl₂ and 80% EtOH. Theprecipitates were dissolved in 20 mM ammonium formate buffer (pH 8.0)and rechromatographed on an anion exchanger.

In this way it is possible to isolate compounds 1 to 6.

EXAMPLE 2 Activation Of Gamma/Delta T-Cells By Enriched MEPIntermediates

Lymphocytes were obtained from the peripheral blood of healthy donors byFicoll density gradient centrifugation. For each test, 2×10⁵ of thecells so obtained were seeded in a volume of 0.2 ml of RPMI-1640 medium(Life Technologies) that was enriched with 25 mM HEPES, 2 mML-glutamine, 0.025 mg/ml of gentamicin, 100 U/ml of human interleukin-2(IL-2) (all from Life Technologies) and 10% human AB serum (Bavarian RedCross). The test fractions were added in various dilutions, andisopentenyl diphosphate (IPP) from Sigma was used in a finalconcentration of 10 μM as a positive control. Incubation was effected inthe incubator with 5% CO₂ at 37° C. After 72 hours the cells wereharvested and analysed in a flow cytometer. In so doing, the expressionof the CD25 activation marker on the surface of V gamma 9⁺ T-cells wasmeasured with the aid of the monoclonal antibodies CD25-PE (B1.49.9), Vgamma 9-FITC (Immu360) and CD3-PC5 (UCHT1) supplied by Beckman-Coulter.

The results showed that compound 1 was approximately 750 times moreactive than IPP, while compound 2 was about 400 times and compounds 3, 5and 6 were about 100 times more active than IPP.

EXAMPLE 3

The synthesis was effected in the manner described in diagram 1:

1. Preparation of compounds Ia to Ic

Compounds Ia and Ib were prepared in an analogous manner to thatdescribed in K. Sato, S. Inoue, Y. Takagi, S. Morii, Bull. Chem. Soc.Jpn., 1976, 49(11), 3351-3351.

The preparation of compound Ic is analogous to that described in H.Kunio, H. Kazushige, Chem. Pharm. Bull., 1994, 42, 4, 786-791.

2. Syntheses of compounds IIa to IIc

Compound IIa was prepared according to current methods which are knownto those skilled in the art, such as have been described in e.g. B.Woodside, Z. Huang, C. Poulter, Org. Synth. 1988, 66, 211-219, startingfrom compound Ib.

Compound IIb was prepared starting from compound Ia. Ia was firstconverted to the corresponding tosylate and then reacted e.g. withtris(tetra-n-butylammonium) hydrogenomethylenediphosphate. The synthesiswas carried out in a manner analogous to that described in WO00/59916and the publications cited therein.

Compound IIc in turn was prepared from compound Ic. The syntheses werecarried out in the manner described in R. C. McClard and T. S. Fujita,J. Am. Chem. Soc., 1987, 109, 5544-5545.

Compound IIc could be obtained in a low yield and was immediatelyhydrolysed in order to obtain compound Mc.

3. Syntheses of compounds IIIa to IIIc

In order to prepare compounds IIIa to IIIc, 500 mg of the correspondingprecursors IIa and IIb were each dissolved in 5 ml of methanol andtreated with 10 mol % of hydrogenation catalyst. Then hydrogen wasintroduced at room temperature and the uptake of hydrogen was measured.After the appropriate amount of hydrogen had been taken up, the mixturewas filtered and the solvent was stripped off. The required productsIIIa and IIIb were obtained with a good degree of purity. Furtherpurification can be achieved by chromatographic methods. Compound IIIcwas obtained from compound IIc. 200 mg of compound IIc were dissolved inabsolute methylene chloride (3 ml) in a heated, argon-flushed flask and10 eq. of trimethylbromosilane were added at 0° C. After stirring forone hour at 0° C., stirring was continued for a further 12 h at roomtemperature. Finally, an aqueous work-up yielded the required productIIIc, which was purified by ion exchange chromatography.

In order to test the activation of gamma/delta T-cells, either theisomerically pure compounds or E/Z mixtures of the compounds were used.

EXAMPLE 4 Purification of Gamma/Delta T-Cell Activating Compounds

Various gamma/delta T-cell activating compounds were isolated fromCoryne-bacterium ammoniagenes. 28 kg of the cell mass were digested witha Dynax mill in 50 mM ammonium formate buffer (pH 8.0). Afterpreabsorption on a hydrophobic polystyrene matrix, the digested materialwas loaded onto an anion exchanger and eluted with a stepped gradient(100, 300, 500 mM ammonium formate, pH 8.0). The 300 mM eluate waspassed through a C-18 matrix and then through a 3 kDa hollow fibrefilter for ultrafiltration. The filtrate was diluted with water to 30 mMammonium formate and loaded once more onto an anion exchanger. Elutionthen took place with a linear gradient of 30 to 500 mM ammonium formate.Individual fractions were tested for their ability to activategamma/delta T-cells. Then some of the active compounds were precipitatedas barium salts by the admixture of 100 mM BaCl₂ and 80% EtOH. Theprecipitates were dissolved in 20 mM ammonium formate buffer (pH 8.0)and rechromatographed on an anion exchanger. In this way it was possibleto isolate compounds 1 to 6, 13 and 14.

EXAMPLE 5 Activation of Gamma/Delta T-Cells by Enriched MEPIntermediates

Lymphocytes were obtained from the peripheral blood of healthy donors byFicoll density gradient centrifugation. For each test, 2×10⁵ of thecells so obtained were seeded in a volume of 0.2 ml of RPMI-1640 medium(Life Technologies) that was enriched with 25 mM HEPES, 2 mML-glutamine, 0.025 mg/ml of gentamicin, 100 U/ml of human interleukin-2(IL-2) (all from Life Technologies) and 10% human AB serum (Bavarian RedCross). The test fractions were added in various dilutions, andisopentenyl diphosphate (IPP) from Sigma was used in a finalconcentration of 10 μM as a positive control. Incubation was effected inthe incubator with 5% CO₂ at 37° C. After 72 hours the cells wereharvested and analysed in a flow cytometer. In so doing, the expressionof the CD25 activation marker on the surface of V gamma 9⁺ T-cells wasmeasured with the aid of the monoclonal antibodies CD25-PE (B 1.49.9), Vgamma 9-FITC (Immu360) and CD3-PC5 (UCHT1) supplied by Beckman-Coulter.

The results showed that compound 9 was approximately 10,000 times moreactive than IPP, compounds 15, 17 and 19 were about 500 times moreactive, compound 10 was around 1000 times more active and compound 12was about 50 times more active than IPP.

1. A method for the treatment of a human or animal disease comprisingthe administration of a compound of formula (I) to a human or animalhaving a disease, wherein said compound of formula (I) is:

in which R₁ is selected from the group consisting of a methyl radical, aformyl radical, substituted or unsubstituted hydroxymethyl radicals andC₀H₂R₃₁, R₃₁ being selected from the group consisting of OH, substitutedor unsubstituted phosphate and substituted or unsubstitutedpyrophosphate and it being impossible for R₃₁ and R₂ to be present inthe molecule simultaneously, R₃₃ is selected from the group consistingof hydrogen, OH, substituted or unsubstituted phosphate and substitutedor unsubstituted pyrophosphate, R₃ is selected from the group consistingof hydrogen, substituted or unsubstituted alkyl having 1 to 26 carbonatoms, substituted or unsubstituted hydroxyalkyl having 1 to 26 carbonatoms, substituted or unsubstituted aryl, substituted or unsubstitutedaralkyl, substituted or unsubstituted alkenyl having 1 to 26 carbonatoms, substituted or unsubstituted alkynyl having 1 to 26 carbon atoms,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclic radicals, substituted or unsubstituted phosphate, a silyl,a nucleoside, a nucleoside monophosphate, diphosphate or triphosphate, adeoxynucleoside, a cation of an organic or inorganic base, a cation ofan organic or inorganic base wherein the metal is from main group I, IIor III of the Periodic Table, ammonium, substituted ammonium, ammoniumcompounds derived from ethylenediamine or amino acids, and OR₃₄, whereinR₃₄ is defined in the same way as R₃, X₂ is defined in the same way asX₁ if a ring is formed between X₂ and C₁ of X₂ is selected from thegroup consisting of —OR₆,

R₇ and R₈ being defined in the same way as R₃₄, and

R₄ being defined in the same way as R₃, Z₁ being defined in the same wayas X₁, and X₃ being defined in the same way as X₁ if it forms a ringwith C₁ and, if it does not form a ring with C₁, being a group

R₅ being defined in the same way as R₃, and Z₂ and X₄, which forms aring with C₁, being defined in the same way as X₁, R₂ is selected fromthe group consisting of hydrogen, OH, alkoxy, phenoxy, benzyloxy,substituted or unsubstituted phosphate and substituted or unsubstitutedpyrophosphate, and X₁ can be oxygen or

Y₁ and Y₂, which can be identical or different, being selected from thegroup consisting of H. OH, halogen, an amino radical, a C₁₋₉-alkoxyradical and a C₁₋₉-alkylthio radical, or together forming an oxo group,and it being possible for a double bond to be present between C₀ and C₁or C₁ and C₂ or C₂ and C₃.
 2. The method according to claim 1, whereinsaid compound has formula (II):

in which a single or double bond is present between C₂ and C₃, R₁ isselected from the group consisting of a methyl radical, a formyl radicaland substituted or unsubstituted hydroxymethyl radicals, R₂ is selectedfrom the group consisting of hydrogen, hydroxyl, alkoxy, phenoxy andbenzyloxy radicals, substituted or unsubstituted phosphate andsubstituted or unsubstituted pyrophosphate, X₁ is oxygen or a group

Y₁ and Y₂, which can be identical or different, being selected from thegroup consisting of H, OH, halogen, amino, a C₁₋₉-alkoxy radical and aC₁₋₉-alkylthio radical, or together forming an oxo group, R₃ is selectedfrom the group consisting of hydrogen, substituted or unsubstitutedalkyl having 1 to 26 carbon atoms, substituted or unsubstitutedhydroxyalkyl having 1 to 26 carbon atoms, substituted or unsubstitutedaryl, substituted or unsubstituted aralkyl, substituted or unsubstitutedalkenyl having 1 to 26 carbon atoms, substituted or unsubstitutedalkynyl having 1 to 26 carbon atoms, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclic radicals,substituted or unsubstituted phosphate, a silyl, a nucleoside, anucleoside monophosphate, diphosphate or triphosphate, adeoxynucleoside, a cation of an organic or inorganic base, especially ametal of main group I, II or III of the Periodic Table, ammonium,substituted ammonium and ammonium compounds derived from ethylenediamineor amino acids, X₂ is defined in the same way as X₁ if a ring is formedbetween X₂ and C₁, and otherwise is

R₄ being defined in the same way as R₃, Z₁ being defined in the same wayas X₁, and X₃ being defined in the same way as X₁ if it forms a ringwith C₁ and, if it does not form a ring with C₁, being a group

R₅ being defined in the same way as R₃, and Z₂ and X₄, which forms aring with C₁, being defined in the same way as X₁.
 3. The methodaccording to claim 2, wherein said compound is of formula (IIA):

in which C₂ and C₃ are joined together by either a single bond or adouble bond, R₁ is a methyl group or a substituted or unsubstitutedhydroxymethyl group, R₂ is selected from the group consisting ofhydrogen, OH, a substituted or unsubstituted phosphate and a substitutedor unsubstituted pyrophosphate, X₁ and X₂ are selected from the groupconsisting of O, CHF, CHCl, CFCl, CH₂, CF₂ and CCl₂, and R₃ is selectedfrom the group consisting of hydrogen, substituted or unsubstitutedphosphate, a nucleoside, a nucleoside monophosphate, diphosphate ortriphosphate, a deoxynucleoside, a cation of an organic or inorganicbase, a cation of an organic or inorganic base wherein the metal is frommain group I, II or III of the Periodic Table, ammonium, substitutedammonium and ammonium compounds derived from ethylenediamine or aminoacids.
 4. The method according to claim 2, wherein said compound is offormula (IIB):

in which C₂ and C₃ are joined together by either a single bond or adouble bond, R₁ is a methyl group or a substituted or unsubstitutedhydroxymethyl group, R₂ is H if R₁ is a substituted or unsubstitutedhydroxymethyl, and is OH, a substituted or unsubstituted phosphate or asubstituted or unsubstituted pyrophosphate if R₁ is a methyl radical,X₁, X₂ and X₃ are selected from the group consisting of O, CHF, CHCl,CFCl, CH₂, CF₂ and CCl₂, and R₃ and R₄ are selected from the groupconsisting of hydrogen, substituted or unsubstituted phosphate, anucleoside, a nucleoside monophosphate, diphosphate or triphosphate, adeoxynucleoside, a cation of an organic or inorganic base, a cation ofan organic or inorganic base wherein the metal is from main group I, IIor III of the Periodic Table, ammonium, substituted ammonium andammonium compounds derived from ethylenediamine or amino acids.
 5. Themethod according to claim 2, wherein said compound is of formula (IIC):

in which there can be a single or double bond between C₂ and C₃, R₁ is amethyl group or a substituted or unsubstituted hydroxymethyl group, R₂is H, OH, a substituted or unsubstituted phosphate or a substituted orunsubstituted pyrophosphate, X₁, X₂, X₃ and X₄ are selected from thegroup consisting of O, CHF, CHCl, CFCl, CH₂, CF₂ and CCl₂, and R₃, R₄and R₅ are selected from the group consisting of hydrogen, substitutedor unsubstituted phosphate, a nucleoside, a nucleoside monophosphate,diphosphate or triphosphate, a deoxynucleoside, a cation of an organicor inorganic base, a cation of an organic or inorganic base wherein themetal is from main group I. II or III of the Periodic Table, ammonium,substituted ammonium and ammonium compounds derived from ethylenediamineor amino acids.
 6. The method according to claim 3, wherein X₁, X₂, X₃and X₄ are oxygen.
 7. The method according to claim 3, wherein R₁ is asubstituted or unsubstituted hydroxymethyl radical, hydroxymethyl, or ahydroxymethyl radical substituted by phosphate, diphosphate ornucleoside diphosphate, and R₂=H.
 8. The method according to claim 3,wherein R₁ is a methyl radical and R₂ is OH, a substituted orunsubstituted phosphate, a substituted pyrophosphate, or a nucleosidediphosphate.
 9. The method according to claim 5, wherein said compoundsare:


10. The method according to claim 1, wherein said compound is of formulaIII:

in which R₃₁ and R₂, which cannot be present in the moleculesimultaneously, are selected from the group consisting of OH,substituted or unsubstituted phosphate and substituted or unsubstitutedpyrophosphate, a double bond being formed between C₁ and C₂ if R₃₁ ispresent in the molecule and a double bond being formed between C₀ and C₁if R₂ is present in the molecule, R₃₃ is selected from the groupconsisting of hydrogen, OH, substituted or unsubstituted phosphate andsubstituted or unsubstituted pyrophosphate, R₃₄ is selected from thegroup consisting of hydrogen, substituted or unsubstituted alkyl having1 to 26 carbon atoms, substituted or unsubstituted hydroxyalkyl having 1to 26 carbon atoms, substituted or unsubstituted aryl, substituted orunsubstituted aralkyl, substituted or unsubstituted alkenyl having 1 to26 carbon atoms, substituted or unsubstituted alkynyl having 1 to 26carbon atoms, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocyclic radicals, substituted or unsubstitutedphosphate, a silyl, a nucleoside, a deoxynucleoside, a nucleosidemonophosphate, diphosphate or triphosphate, a cation of an organic orinorganic base, a cation of an organic or inorganic base wherein themetal is from main group I, II or III of the Periodic Table, ammonium,substituted ammonium and ammonium compounds derived from ethylenediamineor amino acids, X₂ is either —OR₆, R₆ being defined analogously to R₃₄,or can be

R₇ and R₈ being defined in the same way as R₄, and X₁, X₃₂ and X₃₃,which can be identical or different, can be oxygen or a group

Y and Z, which can be identical or different, being selected from thegroup consisting of H, OH, halogen, amino, C₁₋₉-alkoxy andC₁₋₉-alkylthio, or together forming an oxo group.
 11. The methodaccording to claim 10, wherein said compound is of formula (IIIA):

in which R₃₁ and R₂, which cannot be present in the moleculesimultaneously, are selected from the group consisting of OH,substituted or unsubstituted phosphate and substituted or unsubstitutedpyrophosphate, a double bond being formed between C₁ and C₂ if R₃₁ ispresent in the molecule and a double bond being formed analogouslybetween C₀ and C₁ if R₂ is present in the molecule, R₃₄, R₇ and R₈,which can be identical or different, are as defined in claim 1 and X₁,X₃₂ and X₃₃, which can be identical or different, are likewise asdefined in claim
 10. 12. The method according to claim 11, whereinR₃₁=OH and C₀ and C₁ are joined by a double bond.
 13. The methodaccording to claim 11, wherein R₂=OH and C₁ and C₂ are joined by adouble bond.
 14. The method according to claim 10, wherein either R₃₄ orR₆ or R₇ or R₈ is a substituted or unsubstituted phosphate radical. 15.The method according to claim 10, wherein either R₃₁ or R₂ or X₂ is asubstituted or unsubstituted phosphate radical.
 16. The method accordingto claim 10, wherein R₃₄, R₆, R₇ and R₈, which can be identical ordifferent, are hydrogen, a cation of a metal of main group I, II or IIIof the Periodic Table, or substituted or unsubstituted ammonium.
 17. Themethod according to claim 10, wherein X₁ and X₃₂=O.
 18. The methodaccording to claim 10, wherein X₁=CYZ, X₃₂=O and X₃₃=CYZ, Y and Z beingas defined in claim
 10. 19. The method according to claim 10, whereinX₁=O, X₃₂=CYZ and X₃₃=O, Y and Z being as defined in claim
 10. 20. Themethod according to claim 10, wherein X₁, X₃₂ and X₃₃, which can beidentical or different, are selected from the group consisting of CH₂,CHF, CHCl, CFCl, CCl₂ and CF₂.
 21. The method according to claim 10,wherein said compound is selected from the following group:


22. The method according to claim 1, wherein said treatment orprophylaxis is of immune, autoimmune, respiratory diseases or allergiesin humans.
 23. The method according to claim 22, wherein the disease isselected from the group consisting of asthma, Crohn's disease,ulcerative colitis, multiple sclerosis, bone disease, especiallyosteoporosis, and chronic bronchitis, as well as rheumatoid arthritis,Hashimoto's thyroiditis, myasthenia gravis, lupus erythematosus,diabetes mellitus, primary biliary cirrhosis, active chronic hepatitis,adrenalitis/Addison's disease, polymyositis, dermatomyositis, autoimmunehaemolytic anaemia, myocarditis and pericarditis, scleroderma, uveitis,phacouveitis, sympathetic ophthalmia, pemphigus vulgaris, pemphigoid,pernicious anaemia, autoimmune atrophic gastritis, bronchitis, diseasescaused by viruses, bacteria and parasites, benign and malignant tumours,hepatitis C virus infections, and helicobacter eradication therapy onulcers of the gastrointestinal tract.